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DOI: 10.17582/journal.sja/2017.33.1.30.41

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 Sarhad Journal of Agriculture

Research Article

Effective Microorganisms (EM) Co-applied with Organic Wastes and

NPK Stimulate the Growth, Yield and Quality of Spinach (Spinacia
oleracea L.)
Salma Shaheen1*, Mumtaz Khan1, Muhammad Jamil Khan1, Saleem Jilani2, Zarina Bibi1, Muhammad
Munir3 and Mehwish Kiran2

*1Departmentof Soil and Environmental Sciences, Gomal University, Dera Ismail Khan, Pakistan; 2Department
of Horticulture, Gomal University, Dera Ismail Khan, Pakistan; 3Department of Food Sciences, Gomal University,
Dera Ismail Khan, Pakistan.
Abstract | Effective microorganisms (EM) are increasingly being applied to stimulate nutrient cycling and
plant growth. However, information regarding its co-application with organic wastes and chemical fertilizers
on enhancing spinach yield is scarce. A pot experiment was conducted at Gomal University D.I. Khan, Paki-
stan, during 2009-10 to study the changes in soil fertility and spinach growth after the application of EM with
organic wastes and chemical fertilizers. The six treatments were; control (T0), 10 tons (t) ha-1 farm yard manure
(FYM) (T1), 20 t ha-1 pressmud (T2), 0.7 t ha-1 compost (T3), 5 t ha-1 poultry manure (T4) and mixed chemical
fertilizer in the ratio of 100 : 40 : 56 kg ha-1 as N, P and K (T5). Each treatment was applied alone and with
EM. Results showed that EM + pressmud combination significantly improved soil physical and chemical prop-
erties as well as spinach growth compared to other fertilizers with EM. Maximum soil organic matter content
(OM) and K was observed in treatment where pressmud was co-applied with EM. While greater N and P
were found where compost and poultry manure were co-applied with EM respectively. Significant (P < 0.05)
increase in plant height (11%), number of leaves (124%), fresh foliage yield (136%), dry foliage yield (16.4%),
leaf length (20.2%) and leaf area (77.4%) were noted where pressmud + EM was applied as compared to its
corresponding treatment without EM. Maximum Fe, Cu and Mn concentrations were found where pressmud
+ EM was applied. Dietary fiber, vitamin C and crude proteins were also increased in spinach with pressmud +
EM application. It was concluded that EM-inoculated pressmud has higher potential to increases soil fertility
as well as stimulate spinach growth and quality. Therefore, warrants further testing under field conditions.
Received | August 26, 2016; Accepted | January 21, 2017; Published | February 20, 2016
Correspondence | Salma Shaheen, Department of Soil and Environmental Sciences, Gomal University, Dera Ismail Khan, Pakistan; Email:
salmashaheen47@yahoo.com
Citation | Shaheen S., M. Khan., M.J. Khan., S. Jilani., Z. Bibi., M. Munir and M. Kiran. 2017. Effective Microorganisms (EM) co-applied with
organic wastes and NPK stimulate the growth, yield and quality of spinach (Spinacia oleracea l.). Sarhad Journal of Agriculture, 33(1): 30-41.
DOI | http://dx.doi.org/10.17582/journal.sja/2017.33.1.30.41
Keywords | Biomass yield, EM-Technology, Organic manures, Pressmud, Spinach

Introduction dung, poultry manure, pressmud and crop residues,

are locally available and relatively cheap, therefore,

I n many parts of Pakistan, where subsistence farm-

ing is norm, the high cost of chemical fertilizers de-
ters poor farmers to use them on a wide and balanced
increasingly being applied to meet crop nutrient de-
mand (Ahmad et al., 2012). Organic wastes contain
several essential plant nutrients including nitrogen
scale. In such farming system, organic wastes, like cow (N), phosphorus (P), potassium (K), and carbon (C)
March 2017 | Volume 33 | Issue 1 | Page 30

which have the most potential to improve soil physi-
cal, chemical and biological fertility (Schroder, 2005;
Zaman et al., 2004). However, majority of nutrients
in these wastes are found in organic forms which are
reported to get released at different rates (Zaman et
al., 1998; 1999; 2002).
Since, release of nutrients from organic wastes (min-
eralization) is a slow process and carried out by nu- Table 1: Chemical composition of different
merous enzymes and a wide range of microorganisms wastes
(Zaman et al., 1999), therefore, co-application of or-
ganic wastes with effective microorganisms (EM) has
been shown to facilitate release of nutrients such as N
and P (Ahmad et al., 2012; Lack et al., 2013). EM is
a suite of microorganisms which include photosyn- Compost
thetic bacteria, yeasts, fungi and actinomycetes.
In Pakistan, bio-fertilizer products such as EM are now
commercially available. Several studies have reported
positive changes in soil characteristics (Namasivayam,
2010; Chantal, 2013) and plant growth (Zhou et al.,
2009; Talaat, 2014). Application of EM with organic
wastes is reported to improve soil organic matter con-
tent through humification of fresh organic materials;
which then leads to improved soil health and enhance
microbial activities (Valarini et al., 2003). Several
studies have shown that co-application of EM with
organic and chemical fertilizers increased crop yield
(Khaliq et al., 2006). EM applied in combination with
compost increased wheat yield (Hu and Qi, 2013) and
enhanced photosynthesis (Dubey, 2005).
In Pakistan, the efficiency of organic fertilizers is re-
ported to be low probably due to lower soil fertility and
slow release of nutrients from organic nutrient sources
(Tahir et al., 2011; Khan et al., 2012). So, there is a
need to overcome this slow mineralization problem
and provide relatively cheap sources of plant nutrients
to the resource poor farmers. Thus, EM technology
with co-application of organic wastes was introduced
in Pakistan in early 90’s and pioneer research work was
done by Nature Farming Research Centre, University
of Agriculture, Faisalabad (Hussain et al., 1992).
In Pakistan, spinach (Spinacia oleracea L. a member of
Chenopodiaceae family) is among the favorite food
because of its nutritive value such as high Fe and vi-
tamin K contents; however, its per hectare yield of
12588 kg is much lower than those in the developed
countries such as Belgium having per hectare yield of
19700 kg (FAOSTAT, 2012). So, there is a need to
March 2017 | Volume 33 | Issue 1 | Page 31
Sarhad Journal of Agriculture
improve spinach yield to meet the demand of growing
population of Pakistan.
Thus, current study was carried out to assess the ef-
fect of applying organic wastes and chemical fertiliz-
ers with and without EM on soil quality and spinach
growth.
organic
Manures N% P% K%
FYM 2.02 0.58 2.75
Press mud 3.0 3.5 1.8
2.6 0.9 2.0
Poultry 3.1 1.5 2.5
Materials and Methods
A pot experiment was conducted under glasshouse
conditions to assess the effect of co-application of
effective microorganisms (EM) with chemical or or-
ganic fertilizers on soil quality and spinach growth
and quality. Completely randomized design (CRD)
was used with split plot arrangements. There were
six treatments (each with and without EM) i.e. con-
trol (T0), 10 t ha-1 FYM (T1), 20 t ha-1 pressmud (T2),
0.7 t ha-1 compost (T3) and 5 t ha-1 poultry manure
(T4) and the recommended dose of NPK fertilizers
at the rate of 100 : 40 : 56 kg ha-1 (T5). Urea, single
supper phosphate and potassium sulfate were used as
the sources of mixed NPK fertilizers. Farm yard ma-
nure and poultry manure were obtained from nearby
farms while pressmud was obtained from Chashma
Sugar Mills, D. I. Khan. Commercial compost was
purchased from Higo Organic Pvt. Ltd. Organic Fer-
tilizers. Table 1 shows nutrient status of different or-
ganic amendments used in the experiment. Bio-Aab
as source of EM was obtained from EM Foundation,
Faisalabad, Pakistan. Surface soil (0-15 cm) was col-
lected from Indus River basin, sieved through 2mm
mesh and transferred to pots at the rate of 20 Kg soil
pot-1. Pre-sowing analysis of soil indicated 0.9% OM,
440 mg kg-1 N, 4 mg kg-1 P and 47 mg kg-1 K. Or-
ganic manures were saturated with liquid Bio-Aab for
15 days at room temperature (25°C) and applied one
month prior to sowing. Spinach was sown at the seed
rate of 10 g pot-1. Pots were irrigated at regular in-
tervals to keep optimum soil moisture content. Three
spinach harvests were taken and the days taken after
each harvest were recorded. Plant parameters such as
 Sarhad Journal of Agriculture
plant height, number of leaves per plant, fresh and dry Fe and Mn) by using Plasma Mass Spectrometry
foliage yields, and leaf length and leaf area were re- (ICP-MS) as described by Mumtaz et al. (2014). Leaf
corded for each harvest. Leaf length and leaf area were sample (0.5 g) was dried at 80ºC for 72 hours, ground
determined by Graph crude method (Reddy, 2004). to powder with mortar and pestle, and converted
to ash in furnace at 500ºC for 8 hours. The ash was
Soil analysis then digested with 1:1 mixture of HNO3 and HClO4
Post-harvested soil samples were analyzed for their and the extract was analyzed for various elements by
physical and chemical properties. Soil water suspen- ICP-MS. First, the ICP-MS system was calibrated
sion of 1:1 was prepared for pH and electrical con- by using external standards prepared in 1% HNO3.
ductivity measurements (US Salinity Lab Staff, 1954; The standard operating parameters of ICP-MS were
Rhoades, 1996). Organic matter was determined by adjusted to plasma gas flow: 14-16 L min-1, nebulizer
potassium dichromate oxidation method- commonly gas flow: 0.90 – 0.98 L min-1, and sample flow at the
known as Walkley and Black method as described by rate of 1 mL min-1.
(Walkely and Black, 1934). Briefly, 1g air-dried soil
was mixed with 10 mL 1N potassium dichromate and Determination of dietary fiber, vitamin C and crude
20 mL concentrated H2SO4 in 500 mL beaker. After protein
30 minutes, 30 mL distilled water and 10 mL concen- The dietary fiber in spinach was determined by enzy-
trated H3PO4 were added and the mixture was allowed matic-gravimetric method (AOAC, 2000). In short,
to cool. Then, 10-15 drops of diphenylamine indicator powdered spinach samples were gelatinized with heat
were added and mixed thoroughly. Finally, the mixture stable-α amylase and digested with protease and am-
was titrated against 0.5 M ferrous ammonium sulfate yloglucosidase. Soluble and insoluble dietary fibers
till color changed from violet blue to green. Calcula- heat stable-α amylase and digested with protease and
tion for OM was done according to standard equa- amyloglucosidase. Soluble and insoluble dietary fibers
tions. Kjeldahl nitrogen was determined by digesting were precipitated and dried at 105 ºC. Total dietary
and titrating soil sample according to Bremner and fiber was obtained by the sum of soluble and insolu-
Mulvaney (1982). Procedure involved taking 1g dried ble dietary fibers. Vitamin C was determined by using
soil sample in calibrated digestion tube and adding 2, 6-dichloro-indophenol (DCIP) titration method
5g catalyst mixture, few boiling pumice granules and (Tee et al., 1988). Briefly, metaphosphoric acid ex-
15mL concentrated H2SO4 and left overnight. Diges- tracts of spinach leaves were obtained and pH was
tion was carried out in digestion block for 3 hours at adjusted to 1.2. Then, titration was done to determine
370°C and 15 mL distilled water was added for pre- the reducing capacity of extracts with DCIP. Crude
cipitation. Distillation was carried out at steam rate of protein was determined by multiplying total N con-
7-8 mL per minute with saturated H3BO4 and 10N tent by a factor of 6.25 (Fadaei and Salehifar, 2012).
NaOH, followed by titration against 0.01(NH4)2SO4
(pH 5). Extractable phosphorous (P) was determined Statistical analysis
according to the procedure of Olsen et al. (1954) A factorial completely randomized design was fol-
and extractable potassium was determined by flame lowed with three replications. Analysis of variance
photometer (AOAC 2000). Soil saturation percent- (ANOVA) was performed to compare chemical and
organic fertilizers applied with and without EM with
age was calculated from the water loss at 105°C by
respect to spinach yield, and other parameters. Dif-
oven drying of saturated paste to a constant weight:
ferent treatment means were compared using least
significant difference (LSD) test (Steel and Torrie,
1986). All the analyses were performed using Statistix
Where: 9 software.
W1, mass of saturated paste before oven drying; W2,
mass of dried soil paste. Results and Discussion

Determination of micro and macro nutrients in spin-
ach
Fully expanded spinach leaves (3rd and 4th) from the
growing point were analyzed for macro nutrients (N,
P, K, Na, Ca, and Mg) and micro nutrients (Zn, Cu,
March 2017 | Volume 33 | Issue 1 | Page 32
Date of germination and days taken to first harvest
of spinach: Table 2 shows the effect of EM on ger-
mination and days taken for spinach maturity. Plants
grown in the absence of EM took 65 days for first har-
vest while EM-treated plants took 60 days, enhanc-

ing the spinach growth rate by 5 days till harvesting
as compared to non EM treatments. The enhanced
growth rate is attributed to the EM, producing and
inducing the plant growth hormones. Earlier, Kurepin
et al. (2014) reported that bacteria-fungi- and proto-
zoa-based seaweeds extracts act as biostimulators for
inducing different phytohormones like gibberellins,
cytokinines and auxins which regulate plant growth.
Plant height: Physiological traits of spinach during
three harvests under different fertilizers co-applied
with EM are given in Tables 3, 4 and 5. Plant heights
were significantly influenced by the organic and in-
organic fertilizers applied with EM. Significantly
(P<0.05) greater plant to increased plant growth and
crop production. Our results are also supported by
earlier findings of Chantal et al. (2013) who reported
that bio-organic fertilizers with EM significantly in-
creased plant height of flue-cured tobacco.
Number of leaves per plant: Number of leaves per
plant were significantly (P<0.05) affected by differ-
Sarhad Journal of Agriculture
ent organic and chemical fertilizers with and without
EM (Tables 3, 4 and 5). The 1st, 2nd and 3rd spinach
harvests indicated that maximum number of leaves of
59.6, 64.4 and 60.2 were observed where pressmud +
EM was applied followed by FYM (T1) with 32 leaves
per plant that differed from the rest of treatments. The
highest numbers of leaves were in 2nd harvest (138%)
compared to its corresponding non-EM treatment.
Organic amendments with EM were more effective
compared to chemical fertilizer in promoting spinach
growth. This was due to improvement in soil physi-
cal and chemical properties and in addition EM en-
hanced the release of nutrients from organic matter
by rapid decomposition (Ibrahim et al., 2010). Mu-
hammad and Khattak (2009) investigated the effect
of pressmud on plant growth and nutrient status of
maize and reported increased crop yield. Significantly
(P<0.05) greater numbers of leaves per plant were re-
ported in plants where EM was applied indicating the
efficiency of EM. The nutrients in OM are mineralized

Table 2: Date of germination and days taken to first harvest of spinach under different organic and inorganic ferti-
lizers co-applied with EM
Treatment Without EM With EM
Germination dates First harvest time Germination dates First harvest time
T0 12/11/2009 70 days 11/11/2009 68 days
T1 09/11/2009 66 days 07/11/2009 60 days
T2 08/11/2009 65 days 05/11/2009 60 days
T3 09/11/2009 66 days 07/11/2009 60 days
T4 08/11/2009 65 days 06/11/2009 60 days
T5 11/11/2009 68 days 09/11/2009 60 days
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W. EM = without effective microorganisms,
EM = with effective microorganisms (Organic manures socked with Bio-Aab) , Data are means of three replications

Table 3: Physiological traits of spinach in first harvest under different fertilizers co-applied with EM
Treat- Plant Height No. of Leaves Fresh Foliage Dry Foliage Yield Leaf Length Leaf Area
ments (cm) (plant-1) Yield (g pot-1) (g pot-1) (mm) (mm2)
W. EM EM W. EM EM W. EM EM W. EM EM W. EM EM W. EM EM
T0 12.93e 12.93e 11.43e 11.43e 17.33 29.2 2.9 2.9 14.40i 14.40i 24.89 24.89
T1 22.53bcd 30.53ab 15.67cd 27.76b 57.92 103.2 7.69 10.17 24.57de 27.73b 51.19 102.39
T2 28.43ab 32.33a 25.10b 59.56a 95.38 118.67 9.533 10.27 27.37bc 31.33a 98.17 125.98
T3 17.30cde 27.73ab 14.13cde 16.00c 53.67 100.18 6.98 8.30 22.53f 25.40cd 50.82 101.43
T4 12.47e 27.10ab 13.33cde 15.07cde 37.78 53.06 5.37 5.85 20.47g 23.47ef 46.16 78.32
T5 16.30de 24.73bc 11.80de 14.70cde 20.98 70 3.5 3.22 17.47h 19.57gh 37.67 98.72
LSD0.05 8.22 3.88 50.30 3.31 2.18 20.43
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W.EM = without effective microorganisms,
EM = with effective microorganisms (Organic manures socked with Bio-Aab), Data are means of three replications.

March 2017 | Volume 33 | Issue 1 | Page 33

 Sarhad Journal of Agriculture
Table 4: Physiological traits of spinach in second harvest under different fertilizers co-applied with EM
Treat- Plant Height No. of Leaves Fresh Foliage Yield Dry Foliage Yield Leaf Length Leaf Area
ments (cm) (plant-1) (g pot-1) (g pot-1) (mm) (mm2)
W. EM EM W. EM EM W. EM EM W. EM EM W. EM EM W. EM EM
T0 15.43 15.43 13.17e 13.17e 20.27fg 20.27g 3.23cd 3.23d 16.43i 16.43i 26.85e 26.85e
T1 28.66 35.67 26.63c 31.47b 77.36cd 105.25bc 9.89ab 10.63ab 26.57de 29.70b 56.51d 120.07 b
T2 34.98 39.93 27.10bc 64.43a 117.70b 277.85a 11.19ab 13.02a 29.43bc 35.37a 104.68bc 185.69 a
T3 20.86 30.07 19.21d 16.55de 85.18bcd 100.34bc 8.23abcd 9.545bc 24.37ef 27.00cd 53.61 d 110.05bc
T4 14.47 27.73 15.78de 16.01de 63.37de 55.04def 7.23abcd 9.09abc 22.37fg 25.60de 52.45 d 98.12 c
T5 18.87 27.4 15.00de 16.20de 32.71efg 77.72cd 6.15bcd 5.59bcd 19.50h 21.50gh 51.18 d 101.49 c
LSD0.05 10.55 4.67 36.62 6.38 2.44 22.26
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W.EM = without effective microorganisms,
EM = with effective microorganisms (Organic manures socked with Bio-Aab), Data are means of three replications

Table 5: Physiological traits of spinach in third harvest under different fertilizers co-applied with EM
Treat- Plant Height No. of Leaves Fresh Foliage Yield Dry Foliage Yield Leaf Length Leaf Area
ments (cm) (plant-1) (g pot-1) (g pot-1) (mm) (mm2)
W. EM EM W. EM EM W. EM EM W. EM EM W. EM EM W. EM EM
T0 14.93 14.93 13.17c 13.17c 18.77 18.77 3.09 3.09 15.47 15.47 25.47 25.47
T1 26.57 35.33 26.43b 29.30b 63.38 99.30 9.23 10.27 25.77 28.20 46.96bcd 105.44b
T2 32.37 38.70 25.93b 60.15a 116.33 254.83 10.08 10.72 28.70 34.60 92.78 bc 180.21a
T3 19.67 26.63 19.00c 15.52c 80.30 96.77 8.22 8.54 23.50 26.53 46.31bcd 102.00bc
T4 12.47 25.70 14.53c 13.47c 58.48 45.68 6.63 5.94 21.57 24.27 42.69 cd 92.48bc
T5 17.30 22.47 13.00d 15.50 c 27.97 73.03 5.40 4.68 18.53 20.77 40.37 d 97.97bc
LSD0.05 10.03 10.03 43.83 2.38 3.34 62.64
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W.EM = without effective microorganisms,
EM = with effective microorganisms (Organic manures socked with Bio-Aab), Data are means of three replications.

by EM and make them available for plant uptake. cantly (P<0.05) greater fresh foliage yield 136% was
Significantly higher fresh leaf yield of flue-cured to- obtained in 2nd harvest with pressmud + EM as com-
bacco was reported by Chantal et al. (2013) when EM pared to corresponding non-EM treatment. Such in-
was applied with organic fertilizers. Our results are creasing trend of high fresh foliage yield was followed
also supported by the findings of Gorski and Klei- by FYM with EM. Organic wastes improve soil phys-
ber (2010) who reported higher number of leaves ical and chemical fertility through addition of carbo-
in plants sprayed with EM. Similar results were re- naceous and proteinaceous materials which increase
corded by Lack et al. (2013) who reported maximum foliage production through greater availability of nu-
numbers of nods on the stem. They attributed the trients during growth stage. Organic wastes with EM
increased number of leaves to greater photosynthesis also enhance plant growth by producing various plant
and increased crop yield in safflower by the applica- growth regulating hormones and enzymes (Kurepin
tion of crop growth enhancer bacteria. et al., 2014). Effective microorganisms are also in-
volved in the production of antibiotics. Our results are
Fresh foliage yield: Fresh foliage yield of spinach was supported by Arshad et al. (2000), who supplemented
significantly (P<0.05) influenced by organic and in- soil with EM-inoculated farm yard manure and green
organic fertilizers with and without EM (Tables 3, 4 manure with commercially prepared EM (Bio-Aab).
and 5). The 1st, 2nd and 3rd spinach harvest indicated The recorded significant increase in vegetative and re-
improvement in fresh foliage yield. The highest fresh productive growth of wheat plants.
foliage yield 118.7, 277.9 and 254.8 g pot-1, in 1st, 2nd
and 3rd spinach harvests was observed in treatment Dry foliage yield: Dry foliage yield of spinach was
where pressmud + EM was applied, which differed significantly (P<0.05) affected by the application of
significantly from all other treatments. The signifi- organic wastes and chemical fertilizers with EM (Ta-
March 2017 | Volume 33 | Issue 1 | Page 34

bles 3, 4 and 5). The 1 , 2 and 3 spinach harvest pro-
st nd rd
duced highest dry foliage yield of 10.3, 13 and 10.3
g pot-1 where pressmud + EM was applied. EM with
organic sources increased biomass production and fi-
nally increased dry matter content in the crop. Our
results are supported by earlier findings of Lack et al.
(2013). Kurepin et al. (2013, 2014) also revealed that
inoculation with Azospirillum sp. increased grain yield
and shoot dry matter in cereals.
Leaf length: Leaf length is an important yield de-
termining parameter in leafy vegetable. Our results
showed substantial differences among the different
treatments (Tables 3, 4 and 5). The 1st, 2nd and 3rd spin-
ach harvest indicate significantly (P<0.05) maximum
leaf length of 31.3, 35.4 and 34.6 cm in plants receiv-
ing pressmud + EM (T2) which differed from all other
treatments. The statistically (P<0.05) maximum leaf
length of 35.4 cm which is 20.2% of the correspond-
ing non-EM treatment, was found in plants receiving
inoculated pressmud. Khan et al. (2012) reported that
Sarhad Journal of Agriculture
pressmud contained high levels of nitrogen which in-
creased leaf length. Similarly, EM mixture contains
photosynthetic bacteria which may also results in an
increased leaf length. Our results were in line with
those of Chantal et al. (2013) who reported increased
fresh leaf yield of flue-cured tobacco after application
of EM with bio-organic fertilizers.
Leaf area: Leaf area defines the photosynthetic ca-
pacity of a plant. The leaf area of spinach was signifi-
cantly (P<0.05) affected by different organic and in-
organic fertilizers with and without EM (Tables 3, 4
and 5). Results showed maximum leaf areas i.e. 126,
104.7 and 180.2 cm2 in 1st, 2nd and 3rd spinach harvests
respectively with pressmud + EM application. It was
followed by FYM (T1), compost (T3), NPK (T5) and
poultry manure (T4). It could be due to the increased
nitrogen content in organic manures and the nitro-
gen fixation by the EM which then increase leaf area
(Lack et al., 2013). Wang (1999) also found beneficial
effects of pressmud when applied with EM on leaf

Figure 1: Effect of EM and different soil organic fertilizers on micronutrients (Zn, Fe, Cu, and Mn) of spinach
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W. EM = without effective microorganisms,
EM = with effective microorganisms (Organic manures socked with Bio-Aab)
March 2017 | Volume 33 | Issue 1 | Page 35
 Sarhad Journal of Agriculture
photosynthesis and fruit yield of tomato. micronutrient contents have been reported by Gorski
and Kleiber (2010). This increase may be attributed to
Biochemical changes in spinach as affected by differ- increased solubility of the nutrients at low pH: caused
ent nutrient sources co-applied with and without EM by rapid decomposition of the organic substances.

Micro-nutrients status of spinach: The effects of

organic and inorganic fertilizers with and without
EM on the level of micronutrients found in spinach
are shown in Figure 1. The effect of EM on Zn and
Mn was more pronounced compared to Fe and Cu.
Maximum concentrations of Zn, Fe, Cu and Mn were
observed with pressmud + EM application. However,
the highest increase in Zn concentration (66.7%) due
to EM was recorded with poultry manure. Maximum
Fe, Cu and Mn concentrations were found in case of
pressmud + EM application. The minimum values for
all observed micronutrients were obtained in their
respective controls. Previously, such increase in the
Macro-nutrients status of spinach: Figure 2 shows
the effect of organic wastes and NPK fertilizers in the
presence and absence of EM on the levels of macro
nutrients in spinach. Macro-nutrient levels in the
spinach were improved in all treatments receiving
organic fertilizers with EM. Percent improvement
of 8, 2, 6.3, 2, 6.7 and 4 for Na, Ca, Mg, N, P and
K, respectively, in spinach were observed with press-
mud + EM application vs. corresponding pressmud
without EM application. It was followed by FYM,
compost, poultry manure and NPK in the presence
of EM. The lowest values were shown by the control.

Figure 2: Effect of EM and different soil fertilizers on macronutrients (Na, Ca, Mg, N, P and K) of spinach
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W. EM = without effective mi-
croorganisms, EM = with effective microorganisms (Organic manures socked with Bio-Aab)
March 2017 | Volume 33 | Issue 1 | Page 36
 Sarhad Journal of Agriculture

Figure 3: Dietary fiber, vitamin C and crude protein as affected by different soil fertilizers co-applied with EM
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W. EM = without effective mi-
croorganisms, EM = with effective microorganisms (Organic manures socked with Bio-Aab)

Our results are supported by findings of Namasiv-
ayam and Kirithiga (2010). The better results with
EM + pressmud can be attributed to the fact that
pressmud contains approximately 80% moisture and
0.9-1.5% sugars, organic matter, N, P, K, S etc. Both,
sugars and moisture increases population and activ-
ities of effective microorganisms, leading to rapid
nutrient release and subsequent uptake by spinach.
Dietary fiber, vitamin C and crude protein: Sig-
nificant (P<0.05) increase in the dietary fiber (10
%), vitamin C (3.4%) and crude protein (2.5%) in
spinach was observed in the pressmud + EM group
compared to the control treatment and remaining
treatments (Figure 3). Data further showed least
effects of EM in combination with NPK. All the
organic fertilizers in general and specially the press-
mud increased the vitamin C content in spinach
compared to inorganic fertilizers. This may be at-
tributed to the high level of K and carbohydrates
in organic fertilizers which favored the synthesis of
March 2017 | Volume 33 | Issue 1 | Page 37
ascorbic acid in spinach. Since vitamin C production
is tightly linked to carbohydrate metabolism, high
level of nitrogen in inorganic fertilizer increase the
protein production while decrease the carbohydrate
formation therefore vitamin C levels were lower in
inorganic fertilizer (Qureshi et al., 2014).
Effect of EM-inoculated organic wastes and
chemical fertilizers on soil properties: The effect
of co-application of EM with chemical or organ-
ic fertilizers on soil fertility is shown in Figure 4.
Post-harvest soil analyses showed an increase in
the ECe values and a decline in pH. The high-
est increase in ECe value (22.4%) was noted with
pressmud + EM compared to its corresponding
none-EM treatment, while the highest decline
(12%) in pH was observed with pressmud +EM
application compared to treatments without
EM. Decline in soil pH indicates production of
organic acids (humic acid, amino acid, glycine
and cysteine) due to mineralization (amminiza
 Sarhad Journal of Agriculture

Figure 4: Effect of EM and different soil organic fertilizers on electrical conductivity, pH, organic matter, satura-
tion%, nitrogen, phosphorous and potassium in soil after spinach harvesting
T0 = Control, T1 = FYM, T2 = Pressmud, T3 = Compost, T4 = Poultry manure and T5 = NPK, W. EM = without effective microorganisms,
EM = with effective microorganisms (Organic manures socked with Bio-Aab)

tion and ammonification) of organic materials by
heterotrophs and nitrification by autotrophs (Sar-
war et al., 2009). The ECe increased after the ap-
plication of organic manure and EM. During the
decomposition of organic process, microorganisms
produce organic acids which react with soluble
salts present in the soil solution which then lead to
increased soil EC (Sarwar et al., 2008).
further showed improvement in soil fertility as evident
from increased soil OM and saturation percentage.
High OM content in soil (1.25 %) was recorded
with pressmud + EM. The saturation percentage of
35% was observed with pressmud + EM compared
to non-EM treatments. The improvement in key soil
properties, were in the order of pressmud + EM (T2)
> poultry manure + EM (T4) > compost + EM (T3) >
NPK (T5) > control (T1).

Organic and inorganic fertilizer applied with EM EM in combination with organic wastes and chemical
March 2017 | Volume 33 | Issue 1 | Page 38

fertilizers also increased the concentrations of N, P
and K in soil. Results showed that the highest increase
for N (48.5%), P (60%) and K (90%) was observed
where compost + EM, poultry manure + EM and
pressmud + EM were applied compared to none-EM
treatments. This increasing trend may be attributed to
the enhanced microbial activities by EM which led to
nutrients release and availability to plants. Basically,
EM enhances the rapid mineralization of organic
components thus releasing plant nutrients. These
results are supported by findings of Namasivayam and
Kirithiga (2010) who also observed improvement in
soil fertility after EM application.
Conclusion
The findings of our study suggest that EM co-applied
with pressmud, has good potential to increase spinach
yield and quality as well as improve soil fertility and
thus warrants further testing in field conditions under
agro-ecosystems of Pakistan.
Author’s Contribution
This manuscript is the part of corresponding author’s
Ph.D dissertation. Mumtaz Khan contributed in the
analysis of spinach leaves for micro and macro-nu-
trients by plasma mass spectrometry (ICP-MS). The
research work was carried out under the supervision
of Dr. Muhammad Jamil Khan. Salim Jilani provided
the idea of the research. Zarina Bibi assisted in overall
lab work. Muhammad Munir helped in the determi-
nation of dietary fiber and Vitamin C and Mehwish
Kiran contributed in manuscript writing.
References
Ahmad, R., D. Habib, Ehsanullah and A.U. Rehman.
2012. Complimentary effect of organic manures
on the agronomic traits of spring maize. Crop
Environ. 3(1-2): 28-31.
AOAC Association of Official Agricultural
Chemists. 2000. Official methods of analysis,
sodium and potassium in dried milk flame
emission spectrometric method, 17th ed. 2:
63–64.
Arshad, J., A. R. Bajwa, I. Sidiqui and Uzma. 2000.
Nodulation yield and VAM colonization in
vigna mungo (L) in soil with different histories
of EM application. Int. J. Agri. Biol. 101: 1556-
1585.
March 2017 | Volume 33 | Issue 1 | Page 39
Sarhad Journal of Agriculture
Bremner, J.M. and C.S. Mulvaney. 1982. Nitrogen-
Total. In: A.L. Page, R.H. Miller (Eds). Methods
of Soil Analysis. Part 2. 2nd ed. Agron. Monogr.
9. ASA and SSSA, Madison, WI, pp: 595-624. 
Chantal, K., X. Shao, B. Jing, Y. Yuan, M. Hou and L.
Liao. 2013. Effects of effective microorganisms
(EM) and bio-organic fertilizers on the growth
parameters and yield quality of flue-cured
tobacco (Nicotiana tabacum). J. Food Agri.
Environ. 11 (2): 1212-1215.
Dubey, R.S. 2005. Photosynthesis in plants
under stressful conditions. Handbook of
Photosynthesis. 2nd ed . CRC Press, Florida, pp.
479–497.
Fadaei, V. and M. Salehifar. 2012. Some chemical
and functional characteristics of dietary fiber
from five fiber sources. Eur. J. Exp. Biol. 2 (3):
525-528.
FAOSTAT. 2012. http://faostat.fao.org/site/567/
DesktopDefault.aspx?PageID=567#ancor
Gorski, R. and T. Kleiber. 2010. Effect of effective
microorganisms (EM) on nutrient contents in
substrate and development and yield of rose
(Rose x hybrid) and gerbera (Gerbaera jamesonii).
Ecol. Chem. Eng. S.17 (4): 505-513.
Hu, C. and Y. Qi. 2013. Long-term effective
microorganism’s application promote growth
and increase yields and nutrition of wheat in
China. Eur. J. Agron. 46: 63-67. https://doi.
org/10.1016/j.eja.2012.12.003
Hussain, T., S. Muhammad and G. Jilani. 1992.
Prospects of natural agriculture in Pakistan.
Proceedings of the third national congress
of soil science. March 20-22, 1990, Lahore,
Pakistan. Pp 112-122.
Ibrahim, E. A., E.A. Soher, El-Gendy and A. Y.
Ramadan. 2010. Effect of sowing date and some
soil amendments on yield and quality of spinach
(Spinacia oleracea L.). The Sixth International
Conference of Sustainable Agriculture and
Development. Faculty of Agriculture, Fayoum
University, 27-29, 2010. http://www.academia.
edu/8163616
Khaliq, A., M.K. Abassi and T. Hussain. 2006.
Effects of integrated use of organic and
inorganic nutrient sources with effective
microorganisms (EM) on seed cotton yield in
Pakistan. Bioresour. Technol. 97: 967-972.
https://doi.org/10.1016/j.biortech.2005.05.002
Khan, M. J., M. Q. Khan and M. S. Zia. 2012. Sugar
industry press mud as alternate organic fertilizer

source. Int. J. Environ. Waste Manage. 9(1/2):
41-55. https://doi.org/10.1002/jsfa.6545
Kurepin, L. V., M. Zaman and R. P. Pharis. 2014.
Phytohormonal basis for the plant growth
promoting action of naturally occurring
biostimulants. J. Sci. F. Agri. 94(9): 1715-1722.
Kurepin, L. V., J. A. Ozga,M. Zaman and R. P. Pharis.
2013. The Physiology of Plant Hormones in
Cereal, Oilseed and Pulse Crops. Prairie Soils
Crops 6: 7-23.
Lack, S., F. Ghooshchi and H. Hadi. 2013. The
effect of crop growth enhancer bacteria onyield
and yield components of safflower (Carthamus
tinctorius L.) Intl. J. Farm. Alli. Sci. 2(20): 809-
815.
Muhammad, D. and R.A. Khattak. 2009. Growth
and nutrient concentrations of maize in
pressmud treated saline-sodic soils. Soil
Environ. 28(2): 145-155.
Mumtaz K, M.D. Khan, B., Ali, N. Muhammad
and S.J. Zhu. 2014. Differential physiological
and ultrastructural responses of cottonseeds
under Pb toxicity. Pol. J. Environ. Stud. 23(6):
2063-2070.
Namasivayam, S.K.R. 2010. Effect of formulation
of effective microorganism (EM) on post
treatment persistence, microbial density and
soil macronutrients. Rec. Res. Sci. Technol.
2(5): 102-106.
Olsen, S.R., C.V. Cole, F.S. Watanabe and
L.A. Dean. 1954. Estimation of available
phosphorous in soil by extraction with sodium
bicarbonate, USDA Circular. 939.
Qureshi, F., J.A. Wani, U. Bashir, M.A. Malik and
S.A. Mir. 2014. Response of farmyard manure
and inorganic nitrogen on vegetative growth,
leaf yield and quality of kale (Brassica oleracea
L.) in temperate region of Kashmir Valley.
Biolife 2(3):786-791.
Reddy, S.R. 2004. Principles of crop production.
Kalyani publishers Ludhiana – New Delhi –
Noida (UP) Hyderabad - Chennai - Kolkata -
Cuttack.
Rhoades, J.D. 1996. Salinity In: Electrical
conductivity and total dissolved solids. (ed. D.L.
Sparks). Methods of Soil Analysis, 2nd Edition.
Soil Science Society of America, Madison. WI.
pp. 417-436
Sarwar, G., A.R. Naseem and F. Mujeeb. 2009.
Efficiency of various organic materials for
March 2017 | Volume 33 | Issue 1 | Page 40
Sarhad Journal of Agriculture
improving chemical characteristics of normal
soil. Pak. J. Bot. 40(5): 2107-2113.
Sarwar, G., H. Schmeisky, N. Hussain, N.
Muhammad, M. Ibrahimand E. Safdar. 2008.
Improvement of soil physical and chemical
properties with compost application in rice-
wheat cropping system. Pak. J. Bot. 40(1): 275-
282.
Schroder, J. 2005. Revisiting the agronomic benefits
of manure: a correct assessment and exploitation
of its fertilizer value spare the environment.
Bioresour. Technol. 96: 253-261. https://doi.
org/10.1016/j.biortech.2004.05.015
Steel, R.G.D and J.H.Torrie. 1986. Principles and
Procedures of Static. Mc. Graw Hill Book Co
Inc., New York, USA.
Tahir, M., M. Ayub, H.M.R. Javeed, M.Naeem,
H.U. Rehman, M. Waseem and M. Ali. 2011.
Effect of different organic matter on growth
and yield of wheat (Triticum aestivum L.). Pak.
J. Life Soc. Sci. 9(1): 63-66.
Talaat, N.B. 2014. Effective microorganisms
enhance the scavenging capacity of the
ascorbate-glutathione cycle in common bean
(Phaseolus vulgaris L.) plants grown in salty
soils. Plant Physiol. Biochem. 80: 136-143.
https://doi.org/10.1016/j.plaphy.2014.03.035
Tee, E.S., S.I. Young, S.K. Ho and S.S. Mizura.
1988. Determination of vitamin C in fresh
fruits and vegetables using the dye-titration and
microfluorometric methods. Pertanika 11(1):
39-44.
U.S. Salinity Laboratory Staff. 1954. Diagnosis and
improvement of saline and alkali soils. Agric.
Handbook no. 60, USDA. U.S. Government
Printing Office, Washington, DC.
Valarini, P.J., M.C.D. Alvarez., J.M. Gasco., F.
Guerrero and H. Tokeshi. 2003. Assessment
of soil properties by organic matter and EM-
microorganisms incorporation. Rev. Bras. Cienc.
Solo, 27 (3): 519-525. https://doi.org/10.1590/
S0100-06832003000300013
Walkley, A. and I.A. Black. 1934. An examination
of Degtjareff method for determining soil
organic matter, and proposed modification of
the chromic acid titration method. Soil Sci.
37: 29-38. https://doi.org/10.1097/00010694-
193401000-00003
Wang, R. 1999. Effects of organic fertilizer and EM
inoculation on leaf photosynthesis, fruit yield
and quality of tomato plants. J. Nature Farming

Sustain. Environ. 2: 19-126.
Zaman, M., M. Matsushima, S.X. Chang, K. In-
ubushi, M.L. Nguyen, S. Goto, F. Kaneko and T.
Yoneyama. 2004. Nitrogen mineralization, N2O
production and soil microbiological properties
as affected by long-term applications of sewage
sludge composts. Biol. Fertil. Soils 40:101−109.
https://doi.org/10.1007/s00374-004-0746-2
Zaman, M., L.V. Kurepin, W. Catto and R.P.
Pharis. 2015. Enhancing crop yield with the
use of N-based fertilizers co-applied with plant
hormones or growth regulators. J. Sci. F. Agri.
95: (9) 1777-1785. https://doi.org/10.1002/
jsfa.6938
Zaman, M., H.J. Di, K. Sakamoto, S. Goto, H.
Hayashi and K. Inubushi. 2002. Effects of
sewage sludge compost and chemical fertilizer
applications on microbial biomass and N
mineralization rates. Soil Sci. Plant Nutr.
48(2):195−201. https://doi.org/10.1080/00380
March 2017 | Volume 33 | Issue 1 | Page 41
View publication stats
Sarhad Journal of Agriculture
768.2002.10409191
Zaman, M., H.J. Di, K.C. Cameron and C.M.
Frampton. 1999. Gross N mineralization and
nitrification rates and their relationships to
enzyme activities and soil microbial biomass
in soils treated with dairy shed effluent
and ammonium fertilizer at different water
potentials. Biol. Fertil. Soils. 29 (2): 178−186.
https://doi.org/10.1007/s003740050542
Zaman, M., K.C. Cameron, H.J. Di, and M.J.
Noonan. 1998. Nitrogen mineralisation rates
from soil amended with dairy pond waste.
Aust. J. Soil Res. 36: 217−230. https://doi.
org/10.1071/S97048
Zhou, Q., K. Li, X. Jun and L. Bo. 2009. Role
and functions of beneficial microorganisms in
sustainable aquaculture. Bioresour. Technol.
100: 3780-3786. https://doi.org/10.1016/j.
biortech.2008.12.037